Lyophilization is often a preferred formulation for therapeutic materials because the long-term stability of many materials increases in the lyophilized state. However, for plasmid DNA, lyophilized formulations are not the formulations of choice. In most clinical trials using naked (non-complexed plasmid) DNA as a delivery vector, the preferred formulation has been a liquid formulation.
While lyophilized plasmid DNA may be a preferred form of storage, lyophilized formulations for plasmid DNA have been considered to cause a reduction in gene expression efficiency. Lyophilization causes the removal of the hydration sphere around a molecule. For DNA, it appears that there are approximately 20 water molecules per nucleotide pair bound most tightly to DNA that do not form an ice-like structure upon low-temperature cooling. Upon DNA dehydration over hygroscopic salts at 0% relative humidity, only five or six water molecules remain. Thus, lyophilization may increase the stability of DNA under long-term storage, but may also cause some damage upon the initial lyophilization process, potentially through changes in the DNA secondary structure or the concentration of reactive elements such as contaminating metals. Therefore, a potential mechanism for loss of gene expression efficiency of lyophilized plasmid DNA may be through a gross structural change to the plasmid.
In Poxon et al, Pharmaceutical Development and Technology 5:115-122 (2000), the authors demonstrated that lyophilization of a plasmid DNA (pRL-CMV) resulted in a statistically significant loss of transfection efficiency. A biofunctionality assay, measuring transfection activity, demonstrated a loss of more than 75% of plasmid DNA activity after lyophilization as compared to control plasmid that remained in solution. While Poxon et al used carbohydrates to ameliorate the in vitro decreased transfection activity of a non-therapeutic plasmid, pRL-CMV expressing Renilla luciferase, stored in EDTA buffer, Poxon et al did not address the use of lyophilized naked DNA formulations in vivo for disease treatment or prevention.
Therefore, there is a need in the art for a stable lyophilized formulation that will not affect gene expression efficiency. The present invention provides for a lyophilized formulation for plasmid DNA that not only preserves the biological activity of the expressed gene but, in certain instances, is able to enhance biological activity.